Evaluation of the analytical methods of antisense oligonueleotide applicable to the antisense therapy

我们现在正计划对Duchenne肌营养不良症患者进行反义寡核苷酸(反义寡核苷酸，31-mer)的基因治疗。为了监测该反义ODN在注射介质和/或血浆中的浓度，建立了两种分析方法，以获得高灵敏度和良好的可靠性：高效液相色谱法和实时定量聚合酶链式反应方法。反相高效液相色谱法(LC-10A(岛津株式会社))流动相为0.1M三乙胺(pH 8.0)：CH3CN(55：45→95：5)，色谱柱为SG300(资生堂)。该方法在0.50~100μg/ml范围内重现性好，线性关系良好(r^2和0.993)。实时定量聚合酶链式反应方法，反义寡核苷酸的重复性和线性较低(r&lt；0.02)。结果：1.通过改变聚合酶链式反应程序，两种方法均有改善(0.0 5-LOμM，r=0.747)。高效液相色谱法测定反义寡核苷酸在…中的稳定性在几种储存条件下进行了更多的注射研究；遮光或光照，以及4℃、-80℃或室温。将反义寡核苷酸溶解于生理盐水中，在4℃以下遮荫条件下基本稳定5个月(含量大于90%)。反义ODN在生理盐水中的浓度随着光照的增加而降低，并检测到两种降解产物。在生理盐水、蒸馏水、Tris-EDTA缓冲液和5%葡萄糖等介质中，反义ODN几乎稳定到遮光24小时。在测定血浆中反义寡核苷酸浓度时，当血浆浓度为10μg/ml时，加入反义寡核苷酸作为反义寡核苷酸序列的补充，回收率在90%以上。血浆中反义寡核苷酸的最低检测限值为0.5μg/ml，与苯酚提取法相比，该方法简便、简便、灵敏度高，建立了简便、灵敏的反义寡核苷酸检测方法，可用于反义基因治疗中对反义寡核苷酸的稳定性和血药浓度监测。较少

We are now planning to perform the gene therapy with antisense oligonueleotide (antisease ODN, 31-mer) for duchenne muscular dystrophy patients. For the monitoring of this antisense ODN concentrations in injection mediums and/or in plasma, two analytical methods were developed to obtain the high sensitivity and the good reliability ; the high performance liquid chromatography (HPLC) and the real time quantitative PCR methods.1. The reverse phase HPLC method (LC-10A (Shimazu Co. Ltd.)) was consisted from the mobile phase of 0.1 M triethylamine (pH 8.0) : CH_3CN (55 : 45→95 : 5) and ODS column (SG300 (Shiseido Co, Ltd.)). And this method works with high reproducibility at the range of 0.5-100 μg/ml with high linearity (r^2>0.993). For the real time quantitative PCR method, the reproducibility and linearity of antisense ODN with high Tm was much low (r<O.02). By the change of the PCR process, both of them were improved (0.05-lOμM, r=0.747).2. By HPLC method, stability of antisense ODN in … More injection was studied under several storage conditions ; shading or lighting, and 4℃, -80℃, or room temperature. Antisense ODN solved in saline was almost stable for 5 months under shading condition at less than 4℃ (more than 90% contents). In contrast, antisense ODN concentrations in saline were decreased by the light exposure, and two degradation products were detected. In other mediums ; saline, distilled water, Tris-EDTA buffer, and 5% glucose, antisense ODN were almost stable until 24 hrs under shading.3. In the measurement of antisense ODN concentrations in plasma, more than 90% recoveries were obtained at the 10 μg/ml in plasma, when additioning with ODNs complemental to the sequence of antisense ODN. The limited value of antisense ODN in plasma was 0.5 μg/ml. Compared with phenol extraction method, this method was considered to be simple, easy and high sensitive.We established the antisense ODN analytical methods with simple and high sensitivity, which might be applicable to monitor it's stability in injections and plasma concentration for the antisense gene therapy. Less